QIAN SU LAB
  • Home
  • Our research
    • Autophagy and Mitochondria
    • Chromatin DNA Structure
    • Cardiovascular Interactome
    • Single-Mol and Super-Res Tech
  • Publications
  • Our Team
    • Team Members
    • About Peter Su
    • Team Photos
    • Our Collaborators
  • Lab News
  • Resources
    • Facility and Infrastructure
    • Surface Cleaning
    • Antibody Labelling
    • Chromatin DNA Labelling
    • Immunostaining
    • Actin Fluorescent Labelling
    • Motor Protein Purification
    • in vitro Reconstitution
    • Single-molecule Tracking
    • Super-resolution Mapping
    • Codes for SM and SR
  • Contact us
  • Mix Bag
  • Intranet

Chromatin DNA Labeling

Cell Cycle Control and Chromatin DNA Labelling

MATERIALS
Picture
Picture
Picture
METHODS

Day 0 – Sun May 04 201
16:00 
thaw and passage Hela cells from liquid nitrogen.

Day 1 – Mon May 05 2014 11:00 passage cell
In 6‐
well plate, passage the Hela cells 1:2 in fresh medium. 19:00 1st Block (15hrs)
In 6‐
well plate, change fresh medium.
Add Thymidine to final concentration 2mM to Hela cells medium to block the cells.


Day 2 – Tue May 06 2014 10:00 1st Release (10hrs)
To release the cells from S phase, wash cells with 1X DPBS thrice to remove the high concentration of Thymidine, passage Hela cells to coverslip bottomed petri dish to final density 20% ~ 30%.

#VII‐1 #VII‐2 #VII‐3 Hela cells; plate some cells in 6well plate as #VII‐4 dish;

20:00 
2nd Block (15hrs)
In 6
‐well plate and 3 coverslip bottomed petri dishes, change fresh medium.
Add Aphidicolin to final concentration 2ug/ml to Hela cell medium
 
Day 3 - Wed May 07 2014
10:20 make 12ml fresh medium containing 10uM EdU in 15ml tube.
Early-S # VI-1dish Mid-S # VI-2 dish Late-S # VI-3dish
10:30 13:00 15:30
100ul PBS + 25ul FuGene6, incubate 5min at RT. Add 1ul 1mM Atto550-dUTP (Jena Bioscience) to the mix, incubate 20min at 4C freezer, final concentration of the Atto550-dUTP is ~10uM.
11:00 # VI-1,# VI-2,# VI-3, # VI-4 dish To release the cells, wash cells with PBS thrice to remove the aphidicolin, change to 4ml fresh medium (DMEM), then the cells will enter S phase.
Whole Chrom # V-4 dish
11:00 3ml fresh medium + 1ml fresh medium with 10uM EdU. Final EdU is 2.5uM.
Early-S # VI-1dish Mid-S # VI-2 dish Late-S # VI-3dish
11:00 13:30 16:00
Wash cells with PBS twice. Add the ~100ul Atto550-dUTP-FuGene6-PBS Mix to PBS washed cells for 10min at RT. Note: be careful, do not let the cell dry!!!
11:10 13:40 16:10
Fill the dish with 4ml FRESH DMEM. Culture cells at 37C for 0.5 hrs.
 
11:40 14:10 16:40
4ml DMEM medium with EdU, the concentration is 10uM.
12:10 14:40 17:10
Transfer the cell culture dish to B220. Wash cells with PBS briefly. Incubate the cells with Extraction Buffer for 1min to extract the dissociative atto550-dUTP and EdU in the cytoplasm. Rinse once with PBS before the 10min fixation by 4% PFA and 0.1% GA in PBS, followed by 5min incubation of 1mg/ml NaBH4 quenching. Wash with PBS and keep in PBS at 4C.
Note: DO NOT QUENCH !!!
 
Day 3 - Wed May 07 2014
18:00 To permeabilize the cells, incubate the cells with Blocking buffer for 30min at RT
18:30 Remove the Blocking buffer, then wash the cells in each well twice with 1 mL of 5% BSA in PBS. Remove the wash solution.
Picture
18:30 Add 0.5 mL of Click-iT® reaction cocktail to each well containing a coverslip. 
Rock theplate briefly to insure that the reaction cocktail is distributed evenly over the coverslip. Incubate the plate for 30 minutes at room temperature, protected from light.
19:00 Remove the reaction cocktail, then wash each well once with 1 mL of 5% BSA in PBS. Remove the wash solution. 
 
  • blocking and permeabilization with 3% w/v bovine serum albumin (JacksonImmunoResearch Laboratories) and 0.5% v/v Triton X-100 in PBS for 30 min;
  • Note: no need for an extra permeabilization
  • staining for 60min with
    1. Hela – Mouse anti-LaminA/C
  • washing with PBS;
  • staining for 60 min with 
    1. Hela – Mouse anti-LaminA/C – Donkey anti Mouse Atto488
  • washing with PBS;
  • post-fixation in a mixture of 3%formaldehyde and 0.1% glutaraldehyde in PBS for 10 min;
  • washing in PBS.
©Copyright 2020. All Rights Reserved.
Site edited by Dr Qian Peter Su
Last update: 14 Jan 2023
Contact us
[email protected]
​[email protected]
Links to
​UTS | Faculty of Engineering and IT |
School of Biomedical Engineering
​
Proudly powered by Weebly
  • Home
  • Our research
    • Autophagy and Mitochondria
    • Chromatin DNA Structure
    • Cardiovascular Interactome
    • Single-Mol and Super-Res Tech
  • Publications
  • Our Team
    • Team Members
    • About Peter Su
    • Team Photos
    • Our Collaborators
  • Lab News
  • Resources
    • Facility and Infrastructure
    • Surface Cleaning
    • Antibody Labelling
    • Chromatin DNA Labelling
    • Immunostaining
    • Actin Fluorescent Labelling
    • Motor Protein Purification
    • in vitro Reconstitution
    • Single-molecule Tracking
    • Super-resolution Mapping
    • Codes for SM and SR
  • Contact us
  • Mix Bag
  • Intranet